Vcam 1

Human TNFα (recombinant Human TNFα protein, P01375) and mouse TNFα (recombinant mouse TNFα protein, P06804) were purchased from R&D (Minneapolis, MN, USA). The antibodies used for western blotting were as follows: anti-TNF-R1 (C25C1) was from Cell Signaling Technology (Beverly, MA, USA), and anti-β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies used for immunofluorescence staining were VCAM-1 (551146), ICAM-1 (555511), and E-selectin (551145) (BD Biosciences, Franklin Lakes, NJ, USA). The antibodies used for immunohistochemistry staining were TNF-R1 (sc-8436, Santa Cruz Biotechnology, Santa Cruz, CA, USA), VCAM-1 (BA0406, Boster Biological Technology, China), ICAM-1 (WL02268, Wanleibio, China), and E-selectin (abs122144a, Absin, China).

Staining of adhesion molecules on endothelial cells was performed as published previously (40 (link)). Cells were incubated with primary antibodies for E-selectin (R&D Systems), ICAM-1 (R&D Systems), and VCAM-1 (BD Pharmingen) and Alexa Fluor 488-labeled goat anti-mouse IgG (Thermo Fisher Scientific) as a secondary antibody.

For cryosections, tissue was embedded in optimal cutting temperature, frozen on dry ice, and stored at −80°C. Aortic roots were sectioned on a cryostat to generate 10‐ to 15‐μm sections. Cryosections were fixed in acetone for 10 minutes at −20°C, blocked in IHC Tek antibody diluent (IHC World, Ellicott City, MD) for 1 hour at room temperature, and incubated with the indicated antibodies in IHC Tek antibody diluent buffer. Antibodies were phospho‐NFκB‐P65 (1:100; Cell Signaling Technology, Danvers, MA); FN (1:400; Sigma, St. Louis, MO); VCAM‐1 (1:200; BD Biosciences, San Jose, CA); ICAM‐1 (1:200; Biolegend, San Diego, CA); MMP9 (1:200; Abcam, Cambridge, UK); MMP2 (1:300; Millipore, Burlington, MA); CD45 (1:150; BD Pharmingen); CD68 (1:200; Abcam); SMA (1:200; Sigma); F480 (1:200; Serotec, Hercules, CA). Sections were washed 3 times in PBS and incubated with Alexa fluor 598‐conjugated donkey anti‐rabbit or ‐rat secondary antibody (Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Slides were washed with PBS and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). Images were acquired using a Nikon 4 laser confocal microscope. For histology, sections were stained with hematoxylin and eosin, Oil Red, or picrosirius red.